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The Orofacial Formalin Test in the Mouse: A Behavioral Model for Studying Physiology and Modulation of Trigeminal Nociception
The Journal of Pain, Vol 7, No 12 (December), 2006: pp 908-914 Available online at www.sciencedirect.com
The Orofacial Formalin Test in the Mouse: A Behavioral Model for Studying Physiology and Modulation of Trigeminal Nociception Philippe Luccarini,*,† Anne Childeric,*,† Anne-Marie Gaydier,*,† Daniel Voisin,*,† and Radhouane Dallel*,†,‡ *Inserm, Clermont-Ferrand, France. † Université Clermont 1, Clermont-Ferrand, France. ‡ CHU Clermont-Ferrand, Clermont-Ferrand, France.
Abstract: The aim of the current study was to adapt the orofacial formalin pain model previously developed in rats for use in mice and to characterize as fully as possible the behavioral changes in this species. The effects of subcutaneous injection of different formalin concentrations (.5%, 1%, 2%, 4%, and 8%) were examined on the face-rubbing response. In mice, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area. A positive linear relationship between formalin concentration and amplitude of the rubbing activity was observed during the first and second phase of the test with concentration up to 4%. With the highest concentration used (8%), the amplitude of both phases had plateaued. Systemic administration of morphine and paracetamol induced a dose-dependent inhibition of the rubbing behavior during the second phase. Although both paracetamol and morphine inhibited the first phase, a dose-dependent inhibition was found only for morphine. The ED50 value (95% confidence interval) for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg) for morphine. The ED50 values for suppressing the rubbing response during the second phase were 3.52 mg/kg (2.85-4.63 mg/kg) for morphine and 100.66 mg/kg (77.98-139.05 mg/kg) for paracetamol. Heterosegmental nociceptive stimulation evoked by subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin test produced a dose-dependent inhibition of the second phase of the rubbing response. The ED50 values for suppressing the rubbing response during the first and second phases were 9.04 g (1.36-65.13 g) and 0.92 g (0.28-2.99 g), respectively. In conclusion, the mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive orofacial stimulation and the counter-irritation phenomenon and for testing analgesic drugs. Perspective: To further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic “knockout” approach, we require suitable behavioral models in mice. The presented mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive stimulation and the counter-irritation phenomenon and for testing analgesic drugs. © 2006 by the American Pain Society Key words: Rubbing behavior, morphine, paracetamol, capsaicin, pain, counter-irritation.
Received February 7, 2006; Revised April 17, 2006; Accepted April 21, 2006. Supported by grants from INSERM, MENESR, and Fondation Benoit. Address reprint requests to Pr Radhouane Dallel, Inserm E216, Neurobiologie de la douleur trigéminale, Faculté de Chirurgie Dentaire, 11
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Boulevard Charles de Gaulle, 63000 Clermont-Ferrand, France. E-mail: [email protected] 1526-5900/$32.00 © 2006 by the American Pain Society doi:10.1016/j.jpain.2006.04.010
ORIGINAL REPORT/Luccarini et al
T
he orofacial region is one of the most densely innervated, by the trigeminal nerve, areas of the body, which focuses some of the most common acute pains, ie, those accompanying the pathologic states of the teeth and the related structures. It is also the site of frequent chronic post-herpetic neuralgia, migraine, and referred pains. However, the mechanisms underlying these pains are still poorly understood. In particular, there are relatively few behavioral models in laboratory animals dedicated to the study of nociception in the trigeminal region.28,29 In addition, only few analgesic trials have been undergone in trigeminal region. The recent acceleration of basic science studies of pain involving the mouse can largely be attributed to the development of transgenic “knockout” technology in this species only.40 Indeed, a number of transgenic mouse models that display alterations in nociceptive behavior owing to targeted disruption of a gene encoding for a specific receptor, neurotransmitter, or second messenger molecule have recently been described. However, results of these studies have been variously confirmatory, contradictory, and enlightening compared with conventional investigations.40 One reason for these discrepancies is the inconsistent application of behavioral essays of nociception to transgenic mice. This likely results from the small number of laboratories with extensive experience performing behavioral assays of nociception in the mouse40 and from the limited number of models of nociception routinely used in mice. Therefore, to further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic “knockout” approach, we require a more extensive range of suitable behavioral models in mice. We also need to carefully validate behavioral assays of nociception in the mouse, particularly before adapting experimental protocols that were designed for rats. Indeed, the genetic, physiologic, and behavioral differences between rats and mice render such adaptations nontrivial.40 In the case of orofacial pain, there is no behavioral nociceptive test currently used in mice. Some years ago, we adapted the formalin test in the rat14 to assess nociceptive processes in the orofacial region,8 which has since been used with success.29 The aim of the current study was to adapt the orofacial formalin pain model for use in mice and to characterize as fully as possible the behavioral changes evoked in this species. In particular, we carefully studied the relationship between the amount of time the mice spent rubbing their lip and the concentration of the formalin solution, investigated the effects of 2 analgesic drugs, morphine and paracetamol, and investigated the effect of concomitant application of a heterotopic noxious stimulus.
Materials and Methods Animals Adult male NMRI mice weighing 30-35 g (Charles Rivers, Les Oncins, France) were used in these experiments. They were housed in plastic cages (4 per cage) with soft
909 bedding with free access to food and water and were maintained in climate- (23 ⫾ 1°C) and light-controlled (12/12-h dark/light cycle with light on at 8:00 am) protected units (Iffa-Credo, L’Arbresle, France) for at least 1 week before the experiments. Test sessions took place during the light phase between 11:00 AM and 7:00 PM in a quiet room maintained at 23-24°C. The test box had the dimensions of 30 ⫻ 30 ⫻ 15 cm with 3 mirrored sides. Each animal was first placed in this box for a 10-min habituation period to minimize stress. The mice did not have access to food or water during the test. Each mouse was used only once and was killed at the end of the experiment by the administration of a lethal dose of pentobarbital. All procedures were performed in accordance with the European Communities Council Directive 86/ 6609/EEC.
Testing Procedure Nociceptive Effects of Formalin Mice were randomly assigned to 6 groups (8 per group) and received a 10-L subcutaneous injection of diluted formalin or saline into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 0.5%, 1%, 2%, 4%, and 8%. Stock formalin is an aqueous solution of 37% formaldehyde. Formalin was injected subcutaneously through a 27-gauge needle into the center of the right vibrissa pad as quickly as possible, with only minimal animal restraint. Following injection the animals were immediately placed back in the test box for a 45-min observation period. The recording time was divided into 15 blocks of 3 min, and a nociceptive score was determined for each block by measuring the number of seconds that the animals spent grooming the injected area with the ipsilateral fore- or hindpaw. Movements of the ipsilateral forepaw were accompanied by movements of the contralateral forepaw. A videocamera was used to record the grooming response. Analysis of the behavior was made by an investigator who was blinded to the animal’s group assignment.8
Antinociceptive Effects of Morphine and Paracetamol From the protocol described in the preceding, formalin concentration of 4% was chosen as a standard noxious stimulus to evaluate the effects of systemic morphine and paracetamol on the rubbing response. Morphine chlorohydrate and paracetamol were purchased from Sigma Chemical Co (St Louis, MO) and were dissolved in saline (0.9% NaCl solution) and 8% dimethyl sulfoxide (DMSO), respectively. Morphine chlorohydrate and paracetamol were administered subcutaneously into the neck 20 min before formalin. Control animals received saline or 8% DMSO at this time. Morphine was administered at doses of 1.0, 2.0, 4.0, and 8.0 mg/kg (8 per group) and paracetamol was administered at doses of 25, 50, 100, 200, and 400 mg/kg (8 per group).
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Mouse Orofacial Formalin Test
Figure 1. (A) Time course of the face-rubbing activity observed after subcutaneous injection of saline (0%) or different concentrations of formalin into the upper lip. The mean number of seconds that each mouse (8 per concentration) spent rubbing is plotted for each 3-min block over the 45-min postinjection observation period. Effect of increasing concentrations of formalin on (B) the first phase (0-3 min after formalin) and (C) the second phase (15-39 min after formalin) of rubbing activity.
Antinociceptive Effects of Capsaicin: Behavioral Model of Counter-irritation The objective of the experiment was to examine the effect of a distant noxious stimulus on the orofacial formalin test. Capsaicin (Sigma Chemical Co) was used as the counter-irritating stimulus and was dissolved in Endolipid (Braun Medical, Boulogne, France). We examined the effect of different doses of capsaicin (.0, .5, 1, 10, and 50 g; (8 per group)). The mice received 25 L of diluted capsaicin subcutaneously into the back 10 min before formalin (10 L, 4%) into the upper lip.
Testing of Psychomotor Function Changes in motor performance were assessed using the accelerating rotarod test (8500; Ugo Basile, Comerio, Italy), in which mice were required to walk against the motion of a rotating drum, with the speed increasing from 4 to 40 rpm over 5 min. The time (in s) taken to fall off the rotarod was recorded as the latency. Twenty-four hours before drug testing, mice were trained to stay on an accelerating rotarod for 60 s. The next day, rotarod latencies were measured 20 min and 40 min after systemic administration of paracetamol (200 and 400 mg/ kg), morphine (8 mg/kg), vehicle (8% DMSO), or saline (6 per group). In addition, the total time of freezing, defined as a continuous absence of any movements for more than 3 s, was measured during 45 min.
Statistical Analysis Data are presented as mean value ⫾ SEM or ED50 values with 95% confidence intervals. The percentage of antinociceptive effect was calculated as 100 ⫺ (total time of face rubbing in drug assay/total time of face rubbing in saline assay) ⫻ 100. Data were tested for conformity to normality and homogeneity of variance before paramet-
ric analysis. Differences between groups were analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett t test for multiple comparisons between groups (Sigmastat 2.03 for Windows, SPSS, Erkrath, Germany). For correlation between behavioral results and formalin concentration, a Pearson correlation coefficient was used. For all tests the level of significance was set at P ⬍ .05.
Results Effect of Formalin Concentration on the Nociceptive Response The time course of the nociceptive responses to the administration of the different concentrations of formalin is presented on Fig 1. All formalin concentrations from .5% to 8% induced the first phase of the response (Dunnett t test subsequent to ANOVA), which did not last beyond the third minute after injection (Fig 1A). The amplitude of the response reached a maximum at a concentration of 4% (Fig 1B). The second phase of response appeared only with formalin concentrations of 1% and higher (Fig 1A and C) (Dunnett t test subsequent to ANOVA). It started 15 min after formalin injection (block 5) at 4% and 8%. At lower concentrations, the onset of the rubbing activity was delayed until 21 min (block 7) at 2% and 33 min (block 11) at 1%. It also remained significantly lower than the response to 4% and 8% formalin. By the end of the experiment (45 min), the rubbing level had returned to baseline for all concentrations. The graphs shown in Fig 1B and C show that there was a positive linear relationship between the amplitude of the global rubbing activity measured during the first phase (r ⫽ .57) and the second phase (r ⫽ .81) and the
ORIGINAL REPORT/Luccarini et al
911 9.5%, 29.4 ⫾ 10.3%, 36.9 ⫾ 7.2% (P ⬍ .05), and 40.2 ⫾ 9.5% (P ⬍ .05) after administration of 25, 50, 100, and 200 mg/kg, respectively. Although paracetamol inhibited the first phase, a dose-dependent relationship was not found (r ⫽ .37; P ⬎ .05). Systemic administration of paracetamol resulted also in a dose-dependent inhibition of the second phase of the rubbing response (Fig 3). The amplitude of the rubbing response was reduced by 1.4 ⫾ 11.0%, 15.5 ⫾ 11.2%, 41.9 ⫾ 11.0% (P ⬍ .01), and 83.3 ⫾ 7.4% (P ⬍ .001) after administration of 25, 50, 100, and 200 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the second phase was 100.66 mg/kg (77.98-139.05 mg/kg).
Antinociceptive Effects of Capsaicin: Behavioral Model of Counter-irritation Figure 2. Dose-dependent antinociceptive effect of systemic morphine on the rubbing response during the first and second phases of the formalin test (8 per group). Morphine chlorohydrate was administered subcutaneously 20 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
concentration of formalin up to 4%. By the highest concentration (8%), the amplitudes of the responses for both phases had plateaued.
Effect of Formalin Concentration on Freezing Behavior There was no statistically significant evidence of freezing behavior for any of the formalin concentrations used in this study. The duration of freezing was 0 ⫾ 0, 0 ⫾ 0, 0 ⫾ 0, 4.0 ⫾ 2.8, and 1.6 ⫾ 1.6 s after injection of .5%, 1%, 2%, 4%, and 8% of formalin, respectively.
Subcutaneous injection of 50 g capsaicin into the back 10 min before formalin test produced a depression of the first phase of rubbing behavior (Fig 4), whereas lower doses (.5, 1, and 10 g) failed to produce significant effect. The amplitude of the rubbing response was reduced by 68.4 ⫾ 10.7% (P ⬍ .05). The ED50 value for suppressing the rubbing response during the first phase was 9.40 g (1.36-65.13 g). Subcutaneous injection of capsaicin into the back 10 min before formalin test produced a dose-dependent inhibition of the second phase of the rubbing response (Fig 4). The amplitude of the rubbing response was reduced by 24.4 ⫾ 14.1%, 63.7 ⫾ 3.5% (P ⬍ .05), 74.4 ⫾ 7.7% (P ⬍ .01), and 75.4 ⫾ 2.6% (P ⬍ .01) after administration of .5, 1, 10 and 50 g capsaicin, respectively. Thus, for doses beyond 1 g, a ceiling effect was apparent, corresponding to a maximum inhibition of about 75%.
Antinociceptive Effects of Morphine Systemic administration of morphine produced a depression of the first phase of rubbing behavior (Fig 2). The amplitude of the response was reduced by 14.8 ⫾ 8.1%, 47.3 ⫾ 9.6% (P ⬍ .05), 67.4 ⫾ 9.3% (P ⬍ .01), and 90.8 ⫾ 4.2% (P ⬍ .001) after administration of 1, 2, 4 and 8 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg). The administration of morphine also resulted in a dose-dependent inhibition of the second phase of the rubbing response (Fig 2). The amplitude of the response was reduced by 4.6 ⫾ 11.2%, 34.8 ⫾ 8.6% (P ⬍ .05), 48.9 ⫾ 4.7% (P ⬍ .01), and 79.7 ⫾ 3.7% (P ⬍ .001) after administration of 1, 2, 4, and 8 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the second phase was 3.52 mg/kg (2.85-4.63 mg/kg).
Antinociceptive Effects of Paracetamol Systemic administration of paracetamol produced a depression of the first phase of the rubbing behavior (Fig 3). The amplitude of the response was reduced by 11.4 ⫾
Figure 3. Dose-dependent antinociceptive effect of systemic paracetamol on the rubbing response during the first and second phases of the formalin test (8 per group). Paracetamol was administered subcutaneously 20 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
912
Figure 4. Dose-dependent antinociceptive effect of subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin orofacial test. The effect on the rubbing response was measured during the first and second phases of the formalin test (8 per group). Mice received 25 L of diluted capsaicin into the back 10 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
The ED50 value for suppressing the rubbing response during the second phase was .92 g (.28-2.99 g).
Testing of Psychomotor Function Systemic administration of morphine (8 mg/kg) or paracetamol (200 mg/kg) did not affect rotarod performance (Table 1). In contrast, the highest dose of paracetamol (400 mg/kg) induced a great depression of motor and postural activities. Animals treated with 400 mg/kg paracetamol were therefore not included in the study.
Discussion The Orofacial Formalin Test in the Mouse The data reported here indicate that in mice, as in rats, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area (whisker pad, upper lip, and nostril) with the ipsilateral forepaw, sometimes with the hindpaw. The ipsilateral forepaw was often accompanied in its movements by the contralateral forepaw. Furthermore, the behavioral response followed the typical biphasic time course seen in all formalin models.29 A dose-dependent nociceptive effect of formalin was observed during both the first and second phase with concentration up to 4%. Using the paw formalin test in mice, Rosland et al31 obtained a similar positive relationship with concentrations up to 1% and 5% for the early and late phase, respectively. In rats, a positive relationship between formalin concentration and the amplitude of the rubbing activity was observed only for the second phase.8 In the present work, the highest concentration of formalin (8%) did not produce a larger nociceptive behavior
Mouse Orofacial Formalin Test than 4% formalin. Rosland et al31 also noted a similar ceiling effect in mice when paw licking was used as the behavioral nociceptive response. However, in rats the administration of the highest formalin concentrations was associated with a decrease in face rubbing (at 5% and 10%),8 paw licking (15%),38 and paw elevation (5%).9 One explanation for these paradoxical decreases or ceiling effects is that high formalin concentrations could induce other behavioral reactions that may interfere with the primary behavior.15 Indeed, in rats, high concentrations of formalin induce long periods of freezing,15 a behavioral reaction that does not happen in mice. Another explanation is that formalin at high concentration may desensitize peripheral nociceptors, resulting in a lower level of stimulation. Histologic changes actually occur in the mouse paw after injection of 5% formalin.31 As a possible consequence, high formalin concentrations have been reported to produce desensitization of all modalities in the injection site.5
Antinociceptive Effect of Morphine and Paracetamol The present study provides evidence that systemic administration of morphine dose-dependently inhibits formalin-induced orofacial rubbing activity in mice. These findings are consistent with previous demonstration that systemic opioids could produce antinociception in trigeminal24,27,30 and spinal23 animal pain models. The morphine ED50 for the second phase was similar to that found in studies using the paw formalin test18,25,26,32,33 and other chemical pain models23 in mice. It must be noted that the effective doses of morphine in mice are higher that those used in rats24 but still remain much lower than those used in phasic tests, such as the tail flick or hot plate tests.17,35 Systemic administration of paracetamol also induced an inhibitory effect on the nociceptive response in the mouse orofacial formalin test. Paracetamol could produce a dose-dependent inhibition of both phases of the formalin test. Such an effect of paracetamol on both phases has already been reported on the paw formalin test in the mouse6,20 as well as in the rat.2,36 Paracetamol
Table 1. Psychomotor Effects Assessed by the Rotarod Test for Drugs TIME AFTER DRUG ADMINISTRATION TREATMENT
20 MIN
40 MIN
Saline Morphine (8 mg/kg) Vehicle (8% DMSO) Paracetamol (200 mg/kg) Paracetamol (400 mg/kg)
163 ⫾ 4 148 ⫾ 12 155 ⫾ 18 120 ⫾ 19 98 ⫾ 8*
177 ⫾ 14 170 ⫾ 16 172 ⫾ 18 162 ⫾ 27 98 ⫾ 8†
NOTE. Values are presented as mean ⫾ SEM, n ⫽ 6/group. *P ⬍ .05. †P ⬍ .01.
ORIGINAL REPORT/Luccarini et al has also been found to be an effective antinociceptive agent in various other animal models of pain. For instance, systemic administration of paracetamol reduced the biting, scratching, and licking behavior evoked by intrathecal administration of substance P and N-methylD-aspartate1 and induced a dose-dependent antinociception in rats submitted to a mechanical noxious stimulus.27 In the present study, however, the antinociceptive dose (100 mg/kg for both phases) was lower than those described in the literature. The antinociceptive effect of paracetamol was found at doses of 200 mg/kg for both phases in the mouse6,20 and at doses of 300-400 mg/kg for the early phase and 100-200 mg/kg for the late phase in the rat.2 Finally, this study provides the first demonstration that administration of a high dose of paracetamol (400 mg/ kg) can induce a depression of motor and postural activities in mice. Previously, Tjolsen et al36 reported that the administration of 400 mg/kg paracetamol could induce a reduced motor activity in the rat. Paracetamol can cause disturbed thermoregulation,16,37 respiratory problems, and tachycardia.3 Furthermore, intraperitoneal administration of 300 mg/kg paracetamol has been shown to cause severe damage to the liver in mice.21 Therefore, administration of doses of paracetamol higher than 200 mg/kg is not recommended in the mouse.
Behavioral Model of Counter-irritation It has long been appreciated that painful stimuli applied to a body surface can produce an inhibition of nociceptive sensations in other body parts, a phenomenon that was named counter-irritation.39 The current results demonstrate that subcutaneous injection of capsaicin into the back can produce a dose-dependent depression of the rubbing behavior produced by formalin injected into the lip. This is in line with recent studies showing that capsaicin injection (25 g) into either the muzzle or the base of the tail attenuated formalin-evoked behavior in rats4 and that intraplantar injection of capsaicin induced an inhibition of the jaw opening reflex in both awake and anesthetized animals.19 Consistent with these data, previous work demonstrated that electrically as well as chemically (formalin) evoked responses of most of spinal12,22 and trigeminal10,13 wide-dynamic-range
913 neurons could be suppressed by remote noxious stimulation. Although Gear et al19 reported that relatively intense nociceptive stimulation (at least 100 g capsaicin) was required to induce this form of antinociception, here, in agreement with Carlton et al,4 the antinociceptive effect of capsaicin on the formalin behavior was observed at a dose of ⱕ25 g. Furthermore, capsaicin exhibited a ceiling effect; larger doses did not produce any further antinociception. One explanation of this ceiling effect could be that capsaicin at high doses desensitizes peripheral nociceptors via mechanisms that may involve VR-1 receptors.34 In humans, administration of a single high dose of capsaicin can elicit desensitization34 and in rats, intradermal or subcutaneous injections of high doses of capsaicin in the excitatory receptive field of spinal7 and trigeminal nociceptive neurons10,11 also reduce neuronal responses mediated by unmyelinated Cfibers. Diffuse noxious inhibitory controls (DNIC) were proposed to provide the neurophysiologic basis for the counter-irritation phenomenon.39 The DNIC are triggered by activation of A-delta or C peripheral fibers. Because capsaicin selectively activates unmyelinated C-fibers and thinly myelinated A-delta fibers, it may trigger DNIC when injected into the back and thus may cause inhibition of the afferent information which triggers the rubbing behavior.
Conclusion In summary, the present study shows that the mouse orofacial formalin test is a valid and reliable model for evaluation of orofacial nociceptive processing and modulation. The test reflects the time course of noxious stimulation, encoding changes in stimulus intensity within a range of concentrations below 4% during both the early and late phase. It is also sensitive to opioid and nonopioid analgesic drugs and can be modulated by heterotopic chemical noxious stimulation. Finally, when compared with the rat orofacial formalin test, the mouse model appears to encode more accurately the intensity of the nociceptive stimulus during both phases and to be more sensitive to paracetamol but less to morphine.
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12. Dickenson AH, Sullivan AF: Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones. Neurosci Lett 83:207-211, 1987 13. Dickenson AH, Le Bars D, Besson JM: Diffuse noxious inhibitory controls (DNIC). Effects on trigeminal nucleus caudalis neurones in the rat. Brain Res 200:293-305, 1980 14. Dubuisson D, Dennis SG: The formalin test: A quantitative study of the analgesic effects of morphine, meperidine and brain stem stimulation in rats and cats. Pain 45:161-174, 1977 15. Fanselow MS, Baackes MP: Conditioned fear-induced opiate analgesia on the formalin test: Evidence for two aversive motivational systems. Learn Motiv 13:200-221, 1982 16. Feldberg W, Saxena PN: Prostaglandins, endotoxin and lipid A on body temperature in rats. J Physiol 249:601-615, 1975 17. Fischer BD, Carrigan KA, Dykstra LA: Effects of N-methylD-aspartate receptor antagonists on acute morphine-induced and L-methadone–induced antinociception in mice. J Pain 6:425-433, 2005 18. Garret C, Carruette A, Fardin V, Moussaoui S, Peyronel JF, Blanchard JC, Laduron PM: Pharmacological properties of a potent and selective nonpeptide substance P antagonist. Proc Natl Acad Sci USA 88:10208-12, 1991 19. Gear RW, Aley KO, Levine JD: Pain-induced analgesia mediated by mesolimbic reward circuits. J Neurosci 19:71757181, 1999 20. Hunskaar S, Fasmer OB, Hole K: Formalin test in mice, a useful technique for evaluating mild analgesics. J Neurosci Methods 14:69-76, 1985 21. James LP, Mayeux PR, Hinson JA: Acetaminophen-induced hepatotoxicity. Drug Metab Dispos 31:1499-506, 2003 22. Le Bars D, Dickenson AH, Besson JM: Diffuse noxious inhibitory controls (DNIC) I. Effects on dorsal horn convergent neurones in the rat. Pain 6:283-304, 1979 23. Le Bars D, Gozariu M, Cadden SW. Animal models of nociception. Pharmacol Rev 53:597-652, 2001 24. Luccarini P, Perrier L, Degoulange C, Gaydier AM, Dallel R: Synergistic antinociceptive effect of amitriptyline and
29. Raboisson P, Dallel R: The orofacial formalin test. Neurosci Biobehav Rev 28:219-226, 2004 30. Ro JY, Harriott A, Crouse U, Capra NF: Innocuous jaw movements increase c-fos expression in trigeminal sensory nuclei produced by masseter muscle inflammation. Pain 104: 539-548, 2003 31. Rosland JH, Tjølsen A, Mæhle B, Hole K: The formalin test in mice: Effect of formalin concentration. Pain 42:235242, 1990 32. Saddi G, Abbott FV:.The formalin test in the mouse: A parametric analysis of scoring properties. Pain 89:53-63, 2000 33. Sufka KJ, Watson GS, Nothdurft RE, Mogil JS: Scoring the mouse formalin test: Validation study. Eur J Pain 2:351358, 1998 34. Szallasi A, Blumberg PM: Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 51:159-211, 1999 35. Tham SM, Angus JA, Tudor EM, Wright CE: Synergistic and additive interactions of the cannabinoid agonist CP55,940 with mu opioid receptor and alpha2-adrenoceptor agonists in acute pain models in mice. Br J Pharmacol 144: 875-884, 2005 36. Tjolsen A, Lund A, Hole K: Antinociceptive effect of paracetamol in rats is partly dependent on spinal serotonergic systems. Eur J Pharmacol 193:193-201, 1991 37. Walker RM, Massey TE, McElligott TF, Racz WJ: Acetaminophen-induced hypothermia, hepatic congestion, and modification by N-acetylcysteine in mice. Toxicol Appl Pharmacol 59:500-507, 1981 38. Wheeler-Aceto H, Cowan A: Naloxone causes apparent antinociception and pronociception simultaneously in the rat paw formalin test. Eur J Pharmacol 236:193-199, 1993 39. Willer JC, Bouhassira D, Le Bars D: Neurophysiological bases of the counter-irritation phenomenon: Diffuse control inhibitors induced by nociceptive stimulation Neurophysiol Clin 29:379-400, 1999 40. Wilson SG, Mogil JS: Measuring pain in the (knockout) mouse: Big challenges in a small mammal. Behav Brain Res 125:65-73, 2001
The Orofacial Formalin Test in the Mouse: A Behavioral Model for Studying Physiology and Modulation of Trigeminal Nociception Philippe Luccarini,*,† Anne Childeric,*,† Anne-Marie Gaydier,*,† Daniel Voisin,*,† and Radhouane Dallel*,†,‡ *Inserm, Clermont-Ferrand, France. † Université Clermont 1, Clermont-Ferrand, France. ‡ CHU Clermont-Ferrand, Clermont-Ferrand, France.
Abstract: The aim of the current study was to adapt the orofacial formalin pain model previously developed in rats for use in mice and to characterize as fully as possible the behavioral changes in this species. The effects of subcutaneous injection of different formalin concentrations (.5%, 1%, 2%, 4%, and 8%) were examined on the face-rubbing response. In mice, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area. A positive linear relationship between formalin concentration and amplitude of the rubbing activity was observed during the first and second phase of the test with concentration up to 4%. With the highest concentration used (8%), the amplitude of both phases had plateaued. Systemic administration of morphine and paracetamol induced a dose-dependent inhibition of the rubbing behavior during the second phase. Although both paracetamol and morphine inhibited the first phase, a dose-dependent inhibition was found only for morphine. The ED50 value (95% confidence interval) for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg) for morphine. The ED50 values for suppressing the rubbing response during the second phase were 3.52 mg/kg (2.85-4.63 mg/kg) for morphine and 100.66 mg/kg (77.98-139.05 mg/kg) for paracetamol. Heterosegmental nociceptive stimulation evoked by subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin test produced a dose-dependent inhibition of the second phase of the rubbing response. The ED50 values for suppressing the rubbing response during the first and second phases were 9.04 g (1.36-65.13 g) and 0.92 g (0.28-2.99 g), respectively. In conclusion, the mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive orofacial stimulation and the counter-irritation phenomenon and for testing analgesic drugs. Perspective: To further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic “knockout” approach, we require suitable behavioral models in mice. The presented mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive stimulation and the counter-irritation phenomenon and for testing analgesic drugs. © 2006 by the American Pain Society Key words: Rubbing behavior, morphine, paracetamol, capsaicin, pain, counter-irritation.
Received February 7, 2006; Revised April 17, 2006; Accepted April 21, 2006. Supported by grants from INSERM, MENESR, and Fondation Benoit. Address reprint requests to Pr Radhouane Dallel, Inserm E216, Neurobiologie de la douleur trigéminale, Faculté de Chirurgie Dentaire, 11
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Boulevard Charles de Gaulle, 63000 Clermont-Ferrand, France. E-mail: [email protected] 1526-5900/$32.00 © 2006 by the American Pain Society doi:10.1016/j.jpain.2006.04.010
ORIGINAL REPORT/Luccarini et al
T
he orofacial region is one of the most densely innervated, by the trigeminal nerve, areas of the body, which focuses some of the most common acute pains, ie, those accompanying the pathologic states of the teeth and the related structures. It is also the site of frequent chronic post-herpetic neuralgia, migraine, and referred pains. However, the mechanisms underlying these pains are still poorly understood. In particular, there are relatively few behavioral models in laboratory animals dedicated to the study of nociception in the trigeminal region.28,29 In addition, only few analgesic trials have been undergone in trigeminal region. The recent acceleration of basic science studies of pain involving the mouse can largely be attributed to the development of transgenic “knockout” technology in this species only.40 Indeed, a number of transgenic mouse models that display alterations in nociceptive behavior owing to targeted disruption of a gene encoding for a specific receptor, neurotransmitter, or second messenger molecule have recently been described. However, results of these studies have been variously confirmatory, contradictory, and enlightening compared with conventional investigations.40 One reason for these discrepancies is the inconsistent application of behavioral essays of nociception to transgenic mice. This likely results from the small number of laboratories with extensive experience performing behavioral assays of nociception in the mouse40 and from the limited number of models of nociception routinely used in mice. Therefore, to further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic “knockout” approach, we require a more extensive range of suitable behavioral models in mice. We also need to carefully validate behavioral assays of nociception in the mouse, particularly before adapting experimental protocols that were designed for rats. Indeed, the genetic, physiologic, and behavioral differences between rats and mice render such adaptations nontrivial.40 In the case of orofacial pain, there is no behavioral nociceptive test currently used in mice. Some years ago, we adapted the formalin test in the rat14 to assess nociceptive processes in the orofacial region,8 which has since been used with success.29 The aim of the current study was to adapt the orofacial formalin pain model for use in mice and to characterize as fully as possible the behavioral changes evoked in this species. In particular, we carefully studied the relationship between the amount of time the mice spent rubbing their lip and the concentration of the formalin solution, investigated the effects of 2 analgesic drugs, morphine and paracetamol, and investigated the effect of concomitant application of a heterotopic noxious stimulus.
Materials and Methods Animals Adult male NMRI mice weighing 30-35 g (Charles Rivers, Les Oncins, France) were used in these experiments. They were housed in plastic cages (4 per cage) with soft
909 bedding with free access to food and water and were maintained in climate- (23 ⫾ 1°C) and light-controlled (12/12-h dark/light cycle with light on at 8:00 am) protected units (Iffa-Credo, L’Arbresle, France) for at least 1 week before the experiments. Test sessions took place during the light phase between 11:00 AM and 7:00 PM in a quiet room maintained at 23-24°C. The test box had the dimensions of 30 ⫻ 30 ⫻ 15 cm with 3 mirrored sides. Each animal was first placed in this box for a 10-min habituation period to minimize stress. The mice did not have access to food or water during the test. Each mouse was used only once and was killed at the end of the experiment by the administration of a lethal dose of pentobarbital. All procedures were performed in accordance with the European Communities Council Directive 86/ 6609/EEC.
Testing Procedure Nociceptive Effects of Formalin Mice were randomly assigned to 6 groups (8 per group) and received a 10-L subcutaneous injection of diluted formalin or saline into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 0.5%, 1%, 2%, 4%, and 8%. Stock formalin is an aqueous solution of 37% formaldehyde. Formalin was injected subcutaneously through a 27-gauge needle into the center of the right vibrissa pad as quickly as possible, with only minimal animal restraint. Following injection the animals were immediately placed back in the test box for a 45-min observation period. The recording time was divided into 15 blocks of 3 min, and a nociceptive score was determined for each block by measuring the number of seconds that the animals spent grooming the injected area with the ipsilateral fore- or hindpaw. Movements of the ipsilateral forepaw were accompanied by movements of the contralateral forepaw. A videocamera was used to record the grooming response. Analysis of the behavior was made by an investigator who was blinded to the animal’s group assignment.8
Antinociceptive Effects of Morphine and Paracetamol From the protocol described in the preceding, formalin concentration of 4% was chosen as a standard noxious stimulus to evaluate the effects of systemic morphine and paracetamol on the rubbing response. Morphine chlorohydrate and paracetamol were purchased from Sigma Chemical Co (St Louis, MO) and were dissolved in saline (0.9% NaCl solution) and 8% dimethyl sulfoxide (DMSO), respectively. Morphine chlorohydrate and paracetamol were administered subcutaneously into the neck 20 min before formalin. Control animals received saline or 8% DMSO at this time. Morphine was administered at doses of 1.0, 2.0, 4.0, and 8.0 mg/kg (8 per group) and paracetamol was administered at doses of 25, 50, 100, 200, and 400 mg/kg (8 per group).
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Mouse Orofacial Formalin Test
Figure 1. (A) Time course of the face-rubbing activity observed after subcutaneous injection of saline (0%) or different concentrations of formalin into the upper lip. The mean number of seconds that each mouse (8 per concentration) spent rubbing is plotted for each 3-min block over the 45-min postinjection observation period. Effect of increasing concentrations of formalin on (B) the first phase (0-3 min after formalin) and (C) the second phase (15-39 min after formalin) of rubbing activity.
Antinociceptive Effects of Capsaicin: Behavioral Model of Counter-irritation The objective of the experiment was to examine the effect of a distant noxious stimulus on the orofacial formalin test. Capsaicin (Sigma Chemical Co) was used as the counter-irritating stimulus and was dissolved in Endolipid (Braun Medical, Boulogne, France). We examined the effect of different doses of capsaicin (.0, .5, 1, 10, and 50 g; (8 per group)). The mice received 25 L of diluted capsaicin subcutaneously into the back 10 min before formalin (10 L, 4%) into the upper lip.
Testing of Psychomotor Function Changes in motor performance were assessed using the accelerating rotarod test (8500; Ugo Basile, Comerio, Italy), in which mice were required to walk against the motion of a rotating drum, with the speed increasing from 4 to 40 rpm over 5 min. The time (in s) taken to fall off the rotarod was recorded as the latency. Twenty-four hours before drug testing, mice were trained to stay on an accelerating rotarod for 60 s. The next day, rotarod latencies were measured 20 min and 40 min after systemic administration of paracetamol (200 and 400 mg/ kg), morphine (8 mg/kg), vehicle (8% DMSO), or saline (6 per group). In addition, the total time of freezing, defined as a continuous absence of any movements for more than 3 s, was measured during 45 min.
Statistical Analysis Data are presented as mean value ⫾ SEM or ED50 values with 95% confidence intervals. The percentage of antinociceptive effect was calculated as 100 ⫺ (total time of face rubbing in drug assay/total time of face rubbing in saline assay) ⫻ 100. Data were tested for conformity to normality and homogeneity of variance before paramet-
ric analysis. Differences between groups were analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett t test for multiple comparisons between groups (Sigmastat 2.03 for Windows, SPSS, Erkrath, Germany). For correlation between behavioral results and formalin concentration, a Pearson correlation coefficient was used. For all tests the level of significance was set at P ⬍ .05.
Results Effect of Formalin Concentration on the Nociceptive Response The time course of the nociceptive responses to the administration of the different concentrations of formalin is presented on Fig 1. All formalin concentrations from .5% to 8% induced the first phase of the response (Dunnett t test subsequent to ANOVA), which did not last beyond the third minute after injection (Fig 1A). The amplitude of the response reached a maximum at a concentration of 4% (Fig 1B). The second phase of response appeared only with formalin concentrations of 1% and higher (Fig 1A and C) (Dunnett t test subsequent to ANOVA). It started 15 min after formalin injection (block 5) at 4% and 8%. At lower concentrations, the onset of the rubbing activity was delayed until 21 min (block 7) at 2% and 33 min (block 11) at 1%. It also remained significantly lower than the response to 4% and 8% formalin. By the end of the experiment (45 min), the rubbing level had returned to baseline for all concentrations. The graphs shown in Fig 1B and C show that there was a positive linear relationship between the amplitude of the global rubbing activity measured during the first phase (r ⫽ .57) and the second phase (r ⫽ .81) and the
ORIGINAL REPORT/Luccarini et al
911 9.5%, 29.4 ⫾ 10.3%, 36.9 ⫾ 7.2% (P ⬍ .05), and 40.2 ⫾ 9.5% (P ⬍ .05) after administration of 25, 50, 100, and 200 mg/kg, respectively. Although paracetamol inhibited the first phase, a dose-dependent relationship was not found (r ⫽ .37; P ⬎ .05). Systemic administration of paracetamol resulted also in a dose-dependent inhibition of the second phase of the rubbing response (Fig 3). The amplitude of the rubbing response was reduced by 1.4 ⫾ 11.0%, 15.5 ⫾ 11.2%, 41.9 ⫾ 11.0% (P ⬍ .01), and 83.3 ⫾ 7.4% (P ⬍ .001) after administration of 25, 50, 100, and 200 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the second phase was 100.66 mg/kg (77.98-139.05 mg/kg).
Antinociceptive Effects of Capsaicin: Behavioral Model of Counter-irritation Figure 2. Dose-dependent antinociceptive effect of systemic morphine on the rubbing response during the first and second phases of the formalin test (8 per group). Morphine chlorohydrate was administered subcutaneously 20 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
concentration of formalin up to 4%. By the highest concentration (8%), the amplitudes of the responses for both phases had plateaued.
Effect of Formalin Concentration on Freezing Behavior There was no statistically significant evidence of freezing behavior for any of the formalin concentrations used in this study. The duration of freezing was 0 ⫾ 0, 0 ⫾ 0, 0 ⫾ 0, 4.0 ⫾ 2.8, and 1.6 ⫾ 1.6 s after injection of .5%, 1%, 2%, 4%, and 8% of formalin, respectively.
Subcutaneous injection of 50 g capsaicin into the back 10 min before formalin test produced a depression of the first phase of rubbing behavior (Fig 4), whereas lower doses (.5, 1, and 10 g) failed to produce significant effect. The amplitude of the rubbing response was reduced by 68.4 ⫾ 10.7% (P ⬍ .05). The ED50 value for suppressing the rubbing response during the first phase was 9.40 g (1.36-65.13 g). Subcutaneous injection of capsaicin into the back 10 min before formalin test produced a dose-dependent inhibition of the second phase of the rubbing response (Fig 4). The amplitude of the rubbing response was reduced by 24.4 ⫾ 14.1%, 63.7 ⫾ 3.5% (P ⬍ .05), 74.4 ⫾ 7.7% (P ⬍ .01), and 75.4 ⫾ 2.6% (P ⬍ .01) after administration of .5, 1, 10 and 50 g capsaicin, respectively. Thus, for doses beyond 1 g, a ceiling effect was apparent, corresponding to a maximum inhibition of about 75%.
Antinociceptive Effects of Morphine Systemic administration of morphine produced a depression of the first phase of rubbing behavior (Fig 2). The amplitude of the response was reduced by 14.8 ⫾ 8.1%, 47.3 ⫾ 9.6% (P ⬍ .05), 67.4 ⫾ 9.3% (P ⬍ .01), and 90.8 ⫾ 4.2% (P ⬍ .001) after administration of 1, 2, 4 and 8 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg). The administration of morphine also resulted in a dose-dependent inhibition of the second phase of the rubbing response (Fig 2). The amplitude of the response was reduced by 4.6 ⫾ 11.2%, 34.8 ⫾ 8.6% (P ⬍ .05), 48.9 ⫾ 4.7% (P ⬍ .01), and 79.7 ⫾ 3.7% (P ⬍ .001) after administration of 1, 2, 4, and 8 mg/kg, respectively. The ED50 value for suppressing the rubbing response during the second phase was 3.52 mg/kg (2.85-4.63 mg/kg).
Antinociceptive Effects of Paracetamol Systemic administration of paracetamol produced a depression of the first phase of the rubbing behavior (Fig 3). The amplitude of the response was reduced by 11.4 ⫾
Figure 3. Dose-dependent antinociceptive effect of systemic paracetamol on the rubbing response during the first and second phases of the formalin test (8 per group). Paracetamol was administered subcutaneously 20 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
912
Figure 4. Dose-dependent antinociceptive effect of subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin orofacial test. The effect on the rubbing response was measured during the first and second phases of the formalin test (8 per group). Mice received 25 L of diluted capsaicin into the back 10 min before formalin injection (10 L, 4%) into the upper lip. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 (Dunnett t test, subsequent to ANOVA).
The ED50 value for suppressing the rubbing response during the second phase was .92 g (.28-2.99 g).
Testing of Psychomotor Function Systemic administration of morphine (8 mg/kg) or paracetamol (200 mg/kg) did not affect rotarod performance (Table 1). In contrast, the highest dose of paracetamol (400 mg/kg) induced a great depression of motor and postural activities. Animals treated with 400 mg/kg paracetamol were therefore not included in the study.
Discussion The Orofacial Formalin Test in the Mouse The data reported here indicate that in mice, as in rats, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area (whisker pad, upper lip, and nostril) with the ipsilateral forepaw, sometimes with the hindpaw. The ipsilateral forepaw was often accompanied in its movements by the contralateral forepaw. Furthermore, the behavioral response followed the typical biphasic time course seen in all formalin models.29 A dose-dependent nociceptive effect of formalin was observed during both the first and second phase with concentration up to 4%. Using the paw formalin test in mice, Rosland et al31 obtained a similar positive relationship with concentrations up to 1% and 5% for the early and late phase, respectively. In rats, a positive relationship between formalin concentration and the amplitude of the rubbing activity was observed only for the second phase.8 In the present work, the highest concentration of formalin (8%) did not produce a larger nociceptive behavior
Mouse Orofacial Formalin Test than 4% formalin. Rosland et al31 also noted a similar ceiling effect in mice when paw licking was used as the behavioral nociceptive response. However, in rats the administration of the highest formalin concentrations was associated with a decrease in face rubbing (at 5% and 10%),8 paw licking (15%),38 and paw elevation (5%).9 One explanation for these paradoxical decreases or ceiling effects is that high formalin concentrations could induce other behavioral reactions that may interfere with the primary behavior.15 Indeed, in rats, high concentrations of formalin induce long periods of freezing,15 a behavioral reaction that does not happen in mice. Another explanation is that formalin at high concentration may desensitize peripheral nociceptors, resulting in a lower level of stimulation. Histologic changes actually occur in the mouse paw after injection of 5% formalin.31 As a possible consequence, high formalin concentrations have been reported to produce desensitization of all modalities in the injection site.5
Antinociceptive Effect of Morphine and Paracetamol The present study provides evidence that systemic administration of morphine dose-dependently inhibits formalin-induced orofacial rubbing activity in mice. These findings are consistent with previous demonstration that systemic opioids could produce antinociception in trigeminal24,27,30 and spinal23 animal pain models. The morphine ED50 for the second phase was similar to that found in studies using the paw formalin test18,25,26,32,33 and other chemical pain models23 in mice. It must be noted that the effective doses of morphine in mice are higher that those used in rats24 but still remain much lower than those used in phasic tests, such as the tail flick or hot plate tests.17,35 Systemic administration of paracetamol also induced an inhibitory effect on the nociceptive response in the mouse orofacial formalin test. Paracetamol could produce a dose-dependent inhibition of both phases of the formalin test. Such an effect of paracetamol on both phases has already been reported on the paw formalin test in the mouse6,20 as well as in the rat.2,36 Paracetamol
Table 1. Psychomotor Effects Assessed by the Rotarod Test for Drugs TIME AFTER DRUG ADMINISTRATION TREATMENT
20 MIN
40 MIN
Saline Morphine (8 mg/kg) Vehicle (8% DMSO) Paracetamol (200 mg/kg) Paracetamol (400 mg/kg)
163 ⫾ 4 148 ⫾ 12 155 ⫾ 18 120 ⫾ 19 98 ⫾ 8*
177 ⫾ 14 170 ⫾ 16 172 ⫾ 18 162 ⫾ 27 98 ⫾ 8†
NOTE. Values are presented as mean ⫾ SEM, n ⫽ 6/group. *P ⬍ .05. †P ⬍ .01.
ORIGINAL REPORT/Luccarini et al has also been found to be an effective antinociceptive agent in various other animal models of pain. For instance, systemic administration of paracetamol reduced the biting, scratching, and licking behavior evoked by intrathecal administration of substance P and N-methylD-aspartate1 and induced a dose-dependent antinociception in rats submitted to a mechanical noxious stimulus.27 In the present study, however, the antinociceptive dose (100 mg/kg for both phases) was lower than those described in the literature. The antinociceptive effect of paracetamol was found at doses of 200 mg/kg for both phases in the mouse6,20 and at doses of 300-400 mg/kg for the early phase and 100-200 mg/kg for the late phase in the rat.2 Finally, this study provides the first demonstration that administration of a high dose of paracetamol (400 mg/ kg) can induce a depression of motor and postural activities in mice. Previously, Tjolsen et al36 reported that the administration of 400 mg/kg paracetamol could induce a reduced motor activity in the rat. Paracetamol can cause disturbed thermoregulation,16,37 respiratory problems, and tachycardia.3 Furthermore, intraperitoneal administration of 300 mg/kg paracetamol has been shown to cause severe damage to the liver in mice.21 Therefore, administration of doses of paracetamol higher than 200 mg/kg is not recommended in the mouse.
Behavioral Model of Counter-irritation It has long been appreciated that painful stimuli applied to a body surface can produce an inhibition of nociceptive sensations in other body parts, a phenomenon that was named counter-irritation.39 The current results demonstrate that subcutaneous injection of capsaicin into the back can produce a dose-dependent depression of the rubbing behavior produced by formalin injected into the lip. This is in line with recent studies showing that capsaicin injection (25 g) into either the muzzle or the base of the tail attenuated formalin-evoked behavior in rats4 and that intraplantar injection of capsaicin induced an inhibition of the jaw opening reflex in both awake and anesthetized animals.19 Consistent with these data, previous work demonstrated that electrically as well as chemically (formalin) evoked responses of most of spinal12,22 and trigeminal10,13 wide-dynamic-range
913 neurons could be suppressed by remote noxious stimulation. Although Gear et al19 reported that relatively intense nociceptive stimulation (at least 100 g capsaicin) was required to induce this form of antinociception, here, in agreement with Carlton et al,4 the antinociceptive effect of capsaicin on the formalin behavior was observed at a dose of ⱕ25 g. Furthermore, capsaicin exhibited a ceiling effect; larger doses did not produce any further antinociception. One explanation of this ceiling effect could be that capsaicin at high doses desensitizes peripheral nociceptors via mechanisms that may involve VR-1 receptors.34 In humans, administration of a single high dose of capsaicin can elicit desensitization34 and in rats, intradermal or subcutaneous injections of high doses of capsaicin in the excitatory receptive field of spinal7 and trigeminal nociceptive neurons10,11 also reduce neuronal responses mediated by unmyelinated Cfibers. Diffuse noxious inhibitory controls (DNIC) were proposed to provide the neurophysiologic basis for the counter-irritation phenomenon.39 The DNIC are triggered by activation of A-delta or C peripheral fibers. Because capsaicin selectively activates unmyelinated C-fibers and thinly myelinated A-delta fibers, it may trigger DNIC when injected into the back and thus may cause inhibition of the afferent information which triggers the rubbing behavior.
Conclusion In summary, the present study shows that the mouse orofacial formalin test is a valid and reliable model for evaluation of orofacial nociceptive processing and modulation. The test reflects the time course of noxious stimulation, encoding changes in stimulus intensity within a range of concentrations below 4% during both the early and late phase. It is also sensitive to opioid and nonopioid analgesic drugs and can be modulated by heterotopic chemical noxious stimulation. Finally, when compared with the rat orofacial formalin test, the mouse model appears to encode more accurately the intensity of the nociceptive stimulus during both phases and to be more sensitive to paracetamol but less to morphine.
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